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• W2046508710 abstract "Article2 September 2002free access A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium Leonard Bosgraaf Leonard Bosgraaf Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Henk Russcher Henk Russcher Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Janet L. Smith Janet L. Smith Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA, 02472-2829 USA Search for more papers by this author Deborah Wessels Deborah Wessels W.M.Keck Dynamic Image Analysis Facility, Department of Biological Sciences, University of Iowa, Iowa City, IA, 52242 USA Search for more papers by this author David R. Soll David R. Soll W.M.Keck Dynamic Image Analysis Facility, Department of Biological Sciences, University of Iowa, Iowa City, IA, 52242 USA Search for more papers by this author Peter J.M. Van Haastert Corresponding Author Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Leonard Bosgraaf Leonard Bosgraaf Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Henk Russcher Henk Russcher Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Janet L. Smith Janet L. Smith Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA, 02472-2829 USA Search for more papers by this author Deborah Wessels Deborah Wessels W.M.Keck Dynamic Image Analysis Facility, Department of Biological Sciences, University of Iowa, Iowa City, IA, 52242 USA Search for more papers by this author David R. Soll David R. Soll W.M.Keck Dynamic Image Analysis Facility, Department of Biological Sciences, University of Iowa, Iowa City, IA, 52242 USA Search for more papers by this author Peter J.M. Van Haastert Corresponding Author Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Search for more papers by this author Author Information Leonard Bosgraaf1, Henk Russcher1, Janet L. Smith2, Deborah Wessels3, David R. Soll3 and Peter J.M. Van Haastert 1 1Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands 2Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA, 02472-2829 USA 3W.M.Keck Dynamic Image Analysis Facility, Department of Biological Sciences, University of Iowa, Iowa City, IA, 52242 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4560-4570https://doi.org/10.1093/emboj/cdf438 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Chemotactic stimulation of Dictyostelium cells results in a transient increase in cGMP levels, and transient phosphorylation of myosin II heavy and regulatory light chains. In Dictyostelium, two guanylyl cyclases and four candidate cGMP-binding proteins (GbpA–GbpD) are implicated in cGMP signalling. GbpA and GbpB are homologous proteins with a Zn2+-hydrolase domain. A double gbpA/gbpB gene disruption leads to a reduction of cGMP-phosphodiesterase activity and a 10-fold increase of basal and stimulated cGMP levels. Chemotaxis in gbpA−B− cells is associated with increased myosin II phosphorylation compared with wild-type cells; formation of lateral pseudopodia is suppressed resulting in enhanced chemotaxis. GbpC is homologous to GbpD, and contains Ras, MAPKKK and Ras-GEF domains. Inactivation of the gbp genes indicates that only GbpC harbours high affinity cGMP-binding activity. Myosin phosphorylation, assembly of myosin in the cytoskeleton as well as chemotaxis are severely impaired in mutants lacking GbpC and GbpD, or mutants lacking both guanylyl cyclases. Thus, a novel cGMP signalling cascade is critical for chemotaxis in Dictyostelium, and plays a major role in myosin II regulation during this process. Introduction cAMP and cGMP are important signalling molecules in prokaryotes and eukaryotes. These molecules are produced by cyclases, degraded by phosphodiesterases (PDEs), and exert their functions by binding to specific proteins. In prokaryotes, cAMP regulates gene expression by binding to the cyclic nucleotide-binding (cNB) domain of catabolic repressor transcription factors (Passner et al., 2000). In eukaryotes, cAMP and cGMP regulate enzyme and channel activity, mainly via cAMP- and cGMP-dependent protein kinases or by direct binding of cGMP to channels (Houslay and Milligan, 1997). Other recently identified targets are proteins of the Epac family; besides a cAMP-binding region, these proteins contain a Rap1 or Ras guanine exchange factor (GEF) domain (de Rooij et al., 1998). In addition, some PDEs contain a GAF domain, which is an unrelated cGMP-binding domain that regulates PDE activity (Francis et al., 2000). cAMP-dependent protein kinase appears to be a universal target, even occurring in primitive eukaryotes. Much less is known about the synthesis and function of cGMP in lower eukaryotes. Yeast seems to lack cGMP signalling, since the genome of Saccharomyces cerevisiae does not appear to encode putative guanylyl cyclases or proteins containing cGMP-binding domains. Guanylyl cyclases have been identified in Paramecium, Tetra hymena and Plasmodium, but the role of cGMP in these organisms is not yet resolved (Linder et al., 1999). In Dictyostelium, cGMP synthesis is stimulated by the chemoattractants cAMP and folic acid, which bind to G-protein-coupled receptors (reviewed by Van Haastert and Kuwayama, 1997). Guanylyl cyclase is activated for only a few seconds, followed by adaptation of signal transduction. The cGMP produced is degraded rapidly, mainly by a cGMP-stimulated cGMP-specific PDE (Van Haastert et al., 1982b). As a consequence of the brief activation of guanylyl cyclases and cGMP stimulation of PDE activity, cGMP accumulation is transient, reaching a maximum at 10 s and returning to basal levels after 30 s. It has been proposed that cGMP mediates chemotaxis by modulating the phosphorylation and localization of myosin II (reviewed by de la Roche and Cote, 2001). Myosin II is activated transiently after chemotactic stimulation by two mechanisms. First, phosphorylation of the regulatory light chain (RLC) increases the ATPase activity of myosin II. Secondly, myosin II is recruited to the cytoskeleton, a process regulated by phosphorylation of myosin heavy chain (MHC) at three threonines located in the coiled-coil tail (see Berlot et al., 1987; Vaillancourt et al., 1988; Egelhoff et al., 1993; de la Roche and Cote, 2001). Cells have a decreased chemotactic index when the gene encoding MHC is disrupted, or replaced by a mutant gene converting the three threonines to alanines, which results in the constitutive overassembly of MHC into the cytoskeleton (Wessels et al., 1988; Egelhoff et al., 1993; Stites et al., 1998). Cells expressing a mutant RLC with an alanine at the phosphorylated serine also show defects in chemotaxis (Zhang et al., 2002). Studies with mutants suggest that cGMP signalling plays a critical role in orchestrating these changes in myosin phosphorylation and localization. In stmF mutants, which make increased amounts of cGMP in response to chemoattractants, MHC and RLC phosphorylation are delayed or enhanced, while in KI-10 mutants, which do not produce cGMP in response to chemoattractants, no changes in RLC and MHC phosphorylation or translocation of myosin to the cytoskeleton are observed (Liu et al., 1993; Liu and Newell, 1994; Dembinsky et al., 1996). However, the relationship between myosin regulation, cGMP formation and chemotaxis is complicated by the fact that the impaired genes of both stmF and KI-10 have not been identified. To understand the function of cGMP in Dictyostelium, it is essential to identify the genes that encode cGMP-metabolizing enzymes and cGMP target proteins. Recently, we characterized two unusual guanylyl cyclases in Dictyostelium, GCA and sGC, that are not related to vertebrate guanylyl cyclases, but are homologous to 12-transmembrane and soluble adenylyl cyclase, respectively (Roelofs et al., 2001a, b). We have also identified four genes that encode proteins with putative cGMP-binding domains, GbpA–GbpD (Goldberg et al., 2002). Here we use gene disruptions to show that GbpA and GbpB encode novel cGMP-stimulated PDEs and are thus involved in controlling the cGMP signal. GbpC and GbpD encode putative cGMP target proteins containing domains homologous to Ras, a protein kinase and Ras-GEF. Mutants defective in guanylyl cyclase activity or cGMP targets show impaired regulation of myosin II, while computer-assisted motion analysis indicates that these mutants are defective in chemotaxis. Results Gene inactivation The genes encoding GbpA–GbpD were disrupted by homologous recombination in the Dictyostelium uracil auxotroph DH1. Four single and two double knock-outs (gbpA and gbpB, and gbpC and gbpD) were made in this strain by using two selection markers, Bsr and pyr5/6. The positions of disruption of the open reading frames (ORFs) are indicated in Figure 1. Knock-out strains were identified by PCR and confirmed by Southern blot analysis (data not shown). In Dictyostelium, the interruption of a gene with a selection marker often leads to the absence of the (truncated) mRNA, probably because it is unstable. This was observed on northern blots for disruption of gbpA, gbpB and gbpD. In contrast, gbpC− null cells show high expression of the truncated mRNA, which may lead to expression of truncated proteins (data not shown). Therefore, for this study, we made a disruption of the gbpC gene resulting in a 2.5 kb deletion at the beginning of the ORF (see Figure 1). The phenotypes of the mutants are described below in the section on chemotaxis. Figure 1.Four unusual putative cGMP-binding proteins in Dictyostelium. Schematic of the topology of GbpA–GbpD. The positions of disruption of the protein sequences in the knock-out cell lines are indicated by asterisks. The bar refers to 100 amino acids. GbpA and GbpB have the same domain topology; a Zn2+-hydrolase (red) and two cNB domains (blue). GbpC and GbpD are also homologues, sharing an N-terminal Ras-GEF-associated domain (light green) a Ras-GEF domain (green), a GRAM domain (dark blue) and two cNB domains (blue). GbpC has additional N-terminal sequence containing leucine-rich repeats (yellow), a Ras (orange), a MAP kinase kinase kinase (pink) and a DEP (turquoise) domain. Download figure Download PowerPoint cGMP-stimulated cGMP-specific phosphodiesterase activity Dictyostelium contains at least two cGMP PDE activities, one from the cloned DdPDE3 (Kuwayama et al., 2001) and a second enzyme that has been characterized biochemically as cGMP PDE that is stimulated by the analogue 8-bromo-cGMP (8bcGMP) (Van Haastert et al., 1982b). GbpA and GbpB contain a Zn2+-hydrolase domain that is distantly related to class II PDEs (Goldberg et al., 2002). To test whether gbpA and/or gbpB encode a PDE, we analysed gbp-null strains developed till the tight aggregate stage when both genes are expressed at high levels. Table I shows the PDE activity measured with [3H]cAMP or [3H]cGMP in the absence or presence of 8bcAMP and 8bcGMP. Wild-type cells contain significant cGMP PDE activity that is stimulated 2- to 3-fold by 8bcGMP, but not by 8bcAMP. Wild-type cells also contain a cAMP PDE activity that is stimulated by both 8bcAMP and 8bcGMP. These enzyme activities are not significantly different in lysates from gbpC−D− cells (data not shown). gbpA− cells show a strong decrease of cGMP PDE activity, and 8bcGMP no longer stimulates the remaining enzyme activity; in contrast, cAMP PDE activity is not changed in gbpA−. On the other hand, the cGMP PDE activity is not strongly reduced in gbpB− cells, in contrast to the significant reduction of cAMP PDE activity. In gbpA−/B− cells, the activity of both cGMP PDE and cAMP PDE is low, and they are not activated by 8bcAMP and 8bcGMP. These results suggest that GbpA and GbpB are PDEs Table 1. PDE activity in Dictyostelium mutants Cell lines PDE activity (pmol/min/mg) cAMP cGMP – 8bcAMP 8bcGMP – 8bcAMP 8bcGMP Wild-type 2.90 ± 0.08 4.07 ± 0.20 3.91 ± 0.13 5.04 ± 0.28 5.44 ± 0.31 11.5 ± 0.59 gbpA− 2.90 ± 0.12 4.36 ± 0.20 3.63 ± 0.14 2.90 ± 0.13 3.42 ± 0.10 3.17 ± 0.06 gbpB− 1.58 ± 0.03 1.64 ± 0.10 1.62 ± 0.10 4.32 ± 0.13 4.51 ± 0.19 11.0 ± 0.41 gbpA−B− 1.62 ± 0.10 1.61 ± 0.06 1.71 ± 0.12 2.51 ± 0.19 2.45 ± 0.12 2.57 ± 0.25 Characterization of GbpA Wild-type − gbpA− <0.15 <0.28 <0.20 2.14 ± 0.31 2.02 ± 0.33 8.31 ± 0.59 gbpB− − gbpA−B− <0.10 <0.11 <0.16 1.80 ± 0.23 2.06 ± 0.23 8.43 ± 0.48 Characterization of GbpB Wild-type − gbpB− 1.32 ± 0.09 2.43 ± 0.21 2.29 ± 0.17 0.72 ± 0.31 0.93 ± 0.36 <0.72 gbpA− − gbpA−B− 1.28 ± 0.16 2.75 ± 0.21 1.92 ± 0.18 0.38 ± 0.22 0.97 ± 0.16 0.60 ± 0.26 PDE activity was measured in the lysates from tight aggregates with 10 nM [3H]cAMP or [3H]cGMP as substrate in the absence or presence of the activators 8bcAMP or 8bcGMP. The data shown are the means and standard error of triplicate determinations from two experiments. The activity of GbpA was deduced by subtraction of the activities of two cell lines that have the same mixture of PDE enzymes except GbpA. The < sign means that the difference in activity is lower than the standard deviation. Dictyostelium contains several PDE activities besides GbpA and GbpB that are still present in the gbpA− and gbpB− cells. GbpA and GbpB may be characterized by calculating the difference of PDE activity between the appropriate cell lines. Thus, GbpA PDE activity is defined as the difference in PDE activity observed in wild-type cells and gbpA− cells, or the difference in activity in gbpB− and gbpA−B− cells (Table I). This demonstrates that GbpA is a cGMP-specific PDE that is stimulated by 8bcGMP but not by 8bcAMP; cAMP is not hydrolysed by GbpA. These properties are essentially identical to those of the cGMP-stimulated cGMP PDE characterized previously (Van Haastert and Van Lookeren Campagne, 1984). GbpB PDE activity is defined as the difference in PDE activity between wild-type and gbpB− cells, or between gbpA− and gbpA−B− cells (Table I). This reveals that GbpB preferentially hydrolyses cAMP while cGMP is hydrolysed at an ∼4-fold lower rate; both 8bcAMP and 8bcGMP stimulate the enzyme. These properties have been confirmed by overexpressing GbpB in gbpA−B− cells (L.Bosgraaf, H.Russcher, H.Snippe, S.Bader, J.Wind and P.J.M.Van Haastert, submitted for publication). Figure 2A reveals the effect of disruption of gbpA and gbpB on cAMP-stimulated cGMP levels in vivo. Wild-type cells show a rapid increase in cGMP levels from a basal level of ∼1 pmol/107 cells to 6 pmol/107 cells 10 s after stimulation; basal levels are recovered after ∼30 s. This cGMP response is nearly identical in gbpB− cells, but is strongly enhanced in gbpA− cells, with basal cGMP levels of 3 pmol/107 cells and the response increased to 15 pmol/107 cells; basal levels are recovered after ∼120 s. The increased cGMP response in vivo is consistent with the reduced cGMP PDE activity in vitro, confirming that gbpA encodes the cGMP-stimulated cGMP PDE activity. Figure 2.cGMP response in gbp-null cells. The cell were starved for 5 h followed by stimulation with 0.1 μM cAMP. Responses were terminated with perchloric acid, and cGMP levels were measured. (A) The symbols refer to wild-type DH1 (filled circles); gbpA− (open triangles); gbpB− (open squares); and gbpA−B− (closed squares). (B) Wild-type DH1 (filled circles); gbpC− (open inverted triangles); gbpD− (open circles); and gbpC−D− (filled inverted triangles). Identical data are presented for wild-type DH1 in (A) and (B). The results shown are the means of triplicate determinations from a typical experiment repeated once. Download figure Download PowerPoint Disruption of gbpB leads to only a slight increase of cGMP levels. However, disruption of gbpB in a gbpA− background results in a very pronounced increase in basal cGMP levels from 3 pmol/107 cells in gbpA− to 12 pmol/107 cells in gbpA−B−. The cAMP-induced cGMP response is also substantially enhanced and prolonged from a maximum of 15 pmol/107 cells at 10 s after stimulation in gbpA− to 40 pmol/107 cells at 30 s after stimulation in gbpA−B−; basal levels were reached after ∼3–4 min. These results confirm the observation that gbpB encodes an enzyme with low cGMP PDE activity that becomes apparent when the much more active GbpA enzyme is deleted. cAMP- and cGMP-binding capacity The cNB domains of GbpA–GbpD are potential cGMP-binding sites, but may also bind cAMP or other compounds. The binding of 10 nM cAMP or cGMP to lysates from different null cell lines is presented in Figure 3. The binding of cAMP to the cytosolic fraction of wild-type cells and the two double-null strains gbpA−B− and gbpC−D− is essentially identical (Figure 3A), indicating that the Gbp proteins do not comprise a large fraction of the observed cAMP-binding capacity. The binding of 10 nM cGMP is not affected much by disruption of gbpA, gbpB or gbpD, whereas disruption of gbpC results in a 80–90% reduction in cGMP binding (Figure 3B). Figure 3.cAMP and cGMP binding to proteins from gbp-null cells. The binding of 10 nM [3H]cAMP (A) or 10 nM [3H]cGMP (B) to the cytosolic fraction of wild-type DH1 and different null cell lines was measured. The means and standard deviations of two (A) or three (B) experiments with triplicate determinations are shown. Download figure Download PowerPoint Analysis of the affinity and number of cGMP-binding sites in different gbp-null mutants is presented as Scatchard plots (Figure 4). The slope in these curves indicates the affinity, whereas the intercept with the abscissa refers to the number of binding sites. Wild-type cells, as well as mutants gbpA−B− and gbpD−, show non-linear Scatchard plots. Curve fitting with a two-component model indicates the presence of ∼200 fmol/mg protein high affinity cGMP-binding sites with a Kd of ∼4 nM, and ∼1200 fmol/mg protein low affinity cGMP-binding sites with a Kd of ∼250 nM. The figure clearly demonstrates that lysates from gbpC− and gbpC−D− cells have lost high affinity cGMP-binding sites. Residual cGMP binding is very low, which complicates its analysis. Curve fitting statistics indicate that data should be fitted according to a one-component model, yielding low affinity cGMP-binding sites with a capacity of ∼1200 fmol/mg protein and a Kd of ∼500 nM (for statistics see legend of Figure 4). Figure 4.Scatchard plot of cGMP binding to proteins from gbp-null cells. The binding of different concentrations of [3H]cGMP to the cytosolic fraction was determined. The symbols refer to wild-type DH1 (filled circles); gbpA−B− (filled squares); gbpC− (open inverted triangles); gbpD− (open circles); and gbpC−D− (filled inverted triangles). Curve fitting with a two-component model indicates two binding forms in wild-type, gbpA−B− and gbpD− showing 200 ± 35 fmol high affinity cGMP-binding sites/mg protein with a Kd of 3.9 ± 1.5 nM and 1165 ± 134 fmol low affinity cGMP-binding sites/mg protein with a Kd of 257 ± 83 nM. Mutant gbpC− and double mutant gbpC−D− show only a low affinity cGMP binding component with 1235 ± 333 fmol cGMP-binding sites/mg protein with a Kd of 557 ± 220 nM. The data points shown are the means of two experiments with triplicate determinations; the estimated binding constants are the means and standard error of the data pooled from three cell lines (wild-type, gbpA−B− and gbpD−) or two cell lines (gbpC− and gbpC−D−). Download figure Download PowerPoint The consequence of gbpC and gbpD disruption on the cAMP-mediated cGMP response is presented in Figure 2B, revealing essentially normal cGMP responses in the gbpC−, gbpD− and gbpC−D− cells. Myosin II phosphorylation and assembly cGMP signalling is implicated in controlling myosin II phosphorylation and localization during chemotaxis, but the mechanism of this regulation is as yet largely unknown. By isolating genetically engineered mutants in guanylyl cyclases (Roelofs and Van Haastert, 2002), cGMP PDEs and cGMP target proteins, we are now able to test the role of cGMP in myosin II regulation more directly. In wild-type cells, cAMP induces the transient phosphorylation of RLC and MHC, yielding maximal values at ∼30 and 90 s after stimulation, respectively. (Figure 5). Quantification of the blots for RLC phosphorylation reveals a 2.41 ± 0.09-fold increase over pre-stimulated levels (Table II). In gbpA−B− cells, which have very high basal and receptor-stimulated cGMP levels, the stimulation of RLC phosphorylation is significantly larger and persists longer than in wild-type cells. In gca−/sgc− cells, which lack cGMP formation, cAMP induces a very small but statistically significant 1.33 ± 0.16-fold increase in RLC phosphorylation; this stimulation shows approximately the same kinetics as in wild-type cells. The RLC phosphorylation response is also very small in gbpC−D− cells, which lack two cGMP target proteins; in addition, basal phosphorylation of RLC in unstimulated cells was consistently lower than in the other strains. Figure 5.cAMP-induced phosphorylation of myosin heavy chain II (MHC) and regulatory light chain (RLC). Starved cells were incubated with [32P]phosphate for 30 min, followed by stimulation with 1 μM cAMP at t = 0 s. Samples were taken at the times indicated and immunoprecipitated with antibodies against RLC and MHC. The immunoprecipitates were analysed by SDS–PAGE and autoradiography. Typical experiments are presented and were repeated at least once. A quantitative analysis of these data is presented in Table II. Download figure Download PowerPoint Table 2. Myosin II modification in Dictyostelium mutants Response Wild-type gca−/sgc− gbpA−B− gbpC−D− RLC phosphorylation Fold increase 2.41 ± 0.09a, a 1.33 ± 0.16a, a 3.16 ± 0.37a, a 1.55 ± 0.16a, a % response 100 23 ± 18b, b 153 ± 47b, b 39 ± 18b, b MHC phosphorylation Fold increase 1.34 ± 0.07a, a 1.15 ± 0.03a, a 1.39 ± 0.05a, a 1.14 ± 0.04a, a % response 100 45 ± 16b, b 115 ± 31 NS 41 ± 18b, b MHC cytoskeleton (% of total) Basal 52.8 ± 5.3 49.4 ± 4.6 NS 26.0 ± 3.7c 37.1 ± 3.7c Stimulated increase 11.2 ± 3.4a, a –7.1 ± 1.2a, a,b, b 25.5 ± 4.3a, a,b, b –7.3 ± 2.5a, a,b, b Phosphorylation data: the radioactivity incorporated into RLC and MHC was quantified using a phosphoimager. The data are presented as fold increase relative to unstimulated samples within the same experiment, using stimulated levels at 30, 60 and 90 s for RLC, and 60, 90 and 180 s for MHC. The percentage response is defined as (fold increase −1 in mutant)/(fold increase −1 in wild-type). The statistical significance was tested with Students t-test. a The fold increase is significantly different from 1; b the response in the mutant is significantly different from the 100% response in wild-type, all at P < 0.05; NS, not significant. Cytoskeleton data: the distribution of MHC between the Triton-insoluble cytoskeleton and the soluble fraction was determined as described in Figure 6. Data are presented as the percentage of total myosin that is present in the cytoskeleton. The stimulated increase refers to the increase observed relative to basal levels in the same experiment. For calculation of the increase, data are taken at 40–120 s for wild-type, 40–300 s for gbpA−B−, and 10–80 s for gca−/sgc− and gbpC−D− cells. The data shown are the means and standard deviations of 2–3 independent experiments. a The stimulated increase is significantly different from 0; b the increase in the mutant is significantly different from the increase seen in wild-type cells c basal levels are significantly different from wild-type; all at P < 0.05. The cAMP stimulation of MHC phosphorylation shows essentially the same characteristics as phosphorylation of RLC, except that MHC phosphorylation attains a maximum at 90 s versus 30 s for RLC phosphorylation. In addition, the MHC phosphorylation response in wild-type cells is rather small; thus, the effects of the mutations are less pronounced than for RLC phosphorylation (see Table II). In the PDE mutant gbpA−B−, the response is prolonged, whereas in the guanylyl cyclase mutant gca−/sgc− and the cGMP target mutant gbpC−/gbpD− the phosphorylation of MHC is significantly less than in wild-type cells. About 50% of the myosin is found in the cytoskeleton of wild-type cells; cAMP induces a small depletion at 10 s which is followed by a transient increase of myosin in the cytoskeleton with a maximum at ∼90 s after stimulation (Figure 6). A similar pattern, with an initial dissociation followed by a slower association of myosin with the cytoskeleton, was observed by Liu and Newell (1991), whereas a transient association without an initial dissociation was observed by Berlot et al. (1987). Basal levels of MHC in the cytoskeleton are much lower in gbpA−B− cells (26 ± 4% in gbpA−B−, compared with 53 ± 5% in wild-type). Addition of cAMP to gbpA−B− cells induces a relatively large increase in myosin in the cytoskeleton, with a maximum approaching the level seen in wild-type cells. In gca−/sgc− and gbpC−D− cells, basal levels of myosin in the cytoskeleton are somewhat lower than in wild-type cells. Addition of cAMP does not lead to the association of myosin in the cytoskeleton; instead a transient depletion between 20 and 80 s after stimulation was observed (Figure 6). Statistical analysis of the data on myosin phosphorylation and recruitment to the cytoskeleton indicates that the differences between the wild-type and mutant strains are significant (Table II). Figure 6.Association of MHC with the Triton-insoluble cytoskeleton. Starved cells were stimulated with 1 μM cAMP at t = 0 s. At the times indicated, 0.5% Triton was added, and lysates were separated into a supernatant and a Triton-insoluble cytoskeleton. The levels of MHC were determined by western blotting using antibody against MHC. The means of two or three experiments are shown. A quantitative analysis of these data is presented in Table II. The symbols refer to wild-type DH1 (filled circles); guanylyl cyclase gca−/sgc− null cells (open triangles); cGMP phosphodiesterase gbpA−B− null cells (filled inverted triangles); and gbpC−D− null cells lacking two cGMP targets (open squares). Download figure Download PowerPoint Chemotaxis of mutants Cell aggregation on filters is as rapid in gbpA−B− double-null cells as in wild-type cells, but is delayed significantly in gbpC−D− and gca−/sgc− cells by ∼3 h (Table III). All strains eventually form fruiting bodies that have approximately the same size as in wild-type cells. The mutants gbpC−D− and gca−/sgc− cells show a reduced chemotactic response towards cAMP as measured with the semi-quantitative small population assay (data not shown). Table 3. Cell aggregation, locomotion and chemotaxis in Dictyostelium mutants Property Wild-type gca−/sgc− gbpA−B− gbpC−D− Aggregation (n = 3) (n = 3) (n = 3) (n = 3) Aggregation time (h) 8.2 ± 1.0 11.5 ± 1.5* 8.3 ± 0.7 NS 11.2 ± 1.3* Locomotion in buffer (n = 23) (n = 13) (n = 23) (n = 19) Speed (μm/min) 8.2 ± 3 3.9 ± 1.1*** 5.6± 1.7** 4.1 ± 1.2*** Roundness (%) 55 ± 14 77 ± 9*** 71 ± 15** 71 ± 15** Direction change (°/min) 35.6 ± 9.4 42.8 ± 7.0* 47.4 ± 8.1*** 52.7 ± 10.7*** Chemotaxis (n = 31) (n = 36) (n = 31) (n = 37) % positive cells 88 61 97 59 Chemotaxis index +0.46 ± 0.29 +0.06 ± 0.22*** +0.61 ± 0.26* +0.05 ± 0.12*** Speed (μm/min) 9.3 ± 4.7 4.5 ± 1.2*** 6.3 ± 3.0** 3.5 ± 1.4*** Roundness (%) 64 ± 8 80 ± 8*** 60 ± 12 NS 77 ± 11*** Direction change (°/min) 24.0 ± 8.6a 39.1 ± 11.2*** 29.6 ± 12.4 NSa 53.2 ± 13.4*** Cells were deposited on filter paper and allowed to develop till the onset of aggregation. For analysis of locomotion in buffer, cells were deposited on a glass support. The chemotactic response of these cells was measured in a chemotaxis chamber with a spatial gradient of cAMP. Cells were videorecorded for 10 min. The perimeter and the centroid of the cells were determined by computer-assisted analysis. This allows calculation of the speed, roundness (ratio of the long and short axis of the cell), direction change and chemotactic index (the net distance moved towards the source of chemoattractant divided by the total distance moved). The data are derived from 3–5 independent experiment; n = the number of cells analysed. The statistical significance with wild-type cells was calculated using a t-test; * P < 0.05; ** P < 0.01; *** P < 0.001; NS, not significant at P > 0.05. a The direction change in a spatial gradient is significantly different from the direction change in buffer. Quantitative data on cell locomotion and chemotaxis were obtained by computer-assisted" @default.
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• W2046508710 date "2002-09-02" @default.
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• W2046508710 title "A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium" @default.
• W2046508710 cites W106546457 @default.
• W2046508710 cites W129159244 @default.
• W2046508710 cites W1493538670 @default.
• W2046508710 cites W1524509448 @default.
• W2046508710 cites W1527689406 @default.
• W2046508710 cites W1885709862 @default.
• W2046508710 cites W198863919 @default.
• W2046508710 cites W1995847977 @default.
• W2046508710 cites W2018796603 @default.
• W2046508710 cites W2028717691 @default.
• W2046508710 cites W2036369875 @default.
• W2046508710 cites W2039628782 @default.
• W2046508710 cites W2043253654 @default.
• W2046508710 cites W2047886675 @default.
• W2046508710 cites W2050497104 @default.
• W2046508710 cites W2057845894 @default.
• W2046508710 cites W2057985347 @default.
• W2046508710 cites W2061902990 @default.
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• W2046508710 cites W2161294085 @default.
• W2046508710 cites W2162608658 @default.
• W2046508710 cites W2170125438 @default.
• W2046508710 cites W2213519779 @default.
• W2046508710 cites W2344556347 @default.
• W2046508710 cites W2417994431 @default.
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